Project Summary/Abstract Mouse Embryonic Stem (ES) cells are derived from the Inner Cell Mass (ICM) just prior to implantation into the uterus. In the embryo, they are pluripotent, i.e. they give rise to all cell types of the adult organism. When maintained in specific culture conditions, ES cells propagate essentially indefinitely as pluripotent cells in vitro, a property called self-renewal. As a result, ES cells are a useful system for understanding the mechanisms that promote pluripotency versus lineage commitment in the embryo and have become essential to studies of embryonic development and regeneration. Many studies have examined the transcriptional, epigenetic, and signaling programs that contribute to ES cell self-renewal. Growing evidence suggests that the ubiquitin- proteasome system (UPS) also reinforces ES cell identity. The UPS consists of a network of ubiquitin ligases, enzymes that add ubiquitin to substrates; deubiquitinases (DUBs), which oppose the actions of ubiquitin ligases; and the proteasome, which degrades ubiquitinated proteins. The contributions of DUBs to pluripotency are still poorly understood, despite several examples of DUBs promoting self-renewal. In preliminary studies, we have found that the DUB Usp9x promotes the open chromatin state and self-renewal capacity of ES cells. We propose to study this protein in mouse ES cells and will test the hypothesis that Usp9x is an important regulator of ES cell identity, promoting self-renewal by deubiquitinating and stabilizing key components of self- renewal pathways. Specifically, we aim to dissect the role(s) of Usp9x in mouse ES cells by (1) analyzing the consequences of Usp9x genetic deletion and mutation on ES cell self renewal and lineage induction (2) and by identifying key substrates and interacting partners. This work aims to shed light on the interplay between the ubiquitin-proteasome system and self-renewal programs. It is highly relevant to studies of molecular mechanisms underlying intellectual and developmental disability and has the potential to offer insight into how USP9X deregulation promotes human developmental disorders, neurological syndromes, and cancer.